The enteric nervous system (ENS) is a complex network of neurons and glia present in the bowel wall and critical for regulating intestinal motility. Abnormal development of the ENS is the underlying cause of Hirschsprung's disease, a congenital intestinal disorder caused by the absence of enteric ganglia, usually in the distal colorectum, and leading to severe intestinal obstruction in newborns. Defects in the Ret and endothelin receptor B (EdnrB) signaling pathways are required for ENS development and are responsible for many cases of Hirschsprung's disease. However, how these key pathways function in vivo to regulate ENS development in the colorectum, and why mutations cause colorectal aganglionosis in humans, is poorly understood. The broad objective of this proposal is to determine the molecular mechanisms that regulate colorectal ENS development in order to identify potential targets for the treatment of Hirschsprung's disease and other congenital intestinal neuropathies. We hypothesize that Ret and EdnrB signaling act coordinately to influence the migration, proliferation, and differentiation of ENS progenitor cells as they cross the cecal and cloacal regions and that this function is critically important for ENS colonization of the colon. To test this hypothesis, we will use the avian embryo to activate and inhibit gene expression in vivo in order to study the role of these signaling pathways. The major advantages of the avian model system are the ease of performing experimental manipulations throughout embryogenesis and the ability to carry out genetic gain- and loss-of-function studies more quickly and at lower cost than in rodents. We propose to use replication-competent retrovirus for gene overexpression, vector-based RNAi for gene silencing, and organ culture assays. Specific Aim I will establish the role of EdnrB signaling on the migration, survival, proliferation, and differentiation of vagal and sacral ENS progenitor cells during formation of the distal intestinal ENS. Specific Aim II will examine the role of Ret signaling in the etiology of colorectal aganglionosis by focusing on the function of this pathway on migration and proliferation of ENS progenitors as they cross the cecal and cloacal regions. Specific Aim III will use an organ culture model of colorectal aganglionosis that we generate by inhibiting EdnrB signaling in the distal intestine. Activators and inhibitors of Ret activity will be added to EdnrB-deficient intestine in order to rescue the aganglionic phenotype by modulating the balance of activity between these two pathways. These experiments will establish the role of Ret and EdnrB signaling in the distal ENS, provide new insights into mechanisms underlying colorectal ENS development, and identify potential targets for the treatment of neurointestinal disorders.